Fig. 7: Complex ceramide synthase Aur1 was disrupted by accumulated ER calcium in csg2Δ cells.

a Upper, schematic diagram of the hypothesis of calcium accumulation in ER caused disruption of enzymes involved in sphingolipid biosynthesis, leading to increased levels of PHS and blockage of autophagy. Lower, schematic diagram of the effects of mutation in Tsc10, Lip1, and Aur1 on autophagy. b Tsc10-GFP, Lip1-GFP, and Aur1-GFP were expressed in WT cells and csg2Δ cells after starvation in SD-N medium and their proteins levels were analyzed. The blots were probed with anti-GFP antibody and Pgk1 was used as a loading control. The samples derive from the same experiment and gels were processed in parallel. c The interaction between Aur1 and Kei1 was weakened by Csg2 deletion. WT cells and csg2Δ cells with expression of Aur1-GFP and Kei1-GFP were collected before and after starvation in SD-N medium and subject to Co-IP assays. The samples derive from the same experiment and gels were processed in parallel. d Aur1-GFP was expressed in WT cells and csg2Δ cells and its protein levels were analyzed after starvation in SD-N medium with EDTA (5 mM) or EGTA (20 mM). The blots were probed with anti-GFP antibody and Pgk1 was used as a loading control. The samples derive from the same experiment and gels were processed in parallel. e Schematic diagram of the integration of ER calcium homeostasis with sphingolipid metabolism and autophagy by calcium channel Csg2. Csg2 is localized at the ER membranes and functions as a calcium release channel maintaining the calcium homeostasis in ER. When Csg2 is absent, abnormally high levels of calcium accumulates in ER to cause the disruption of complex ceramide (IPC) synthase Aur1, which results in increase of bioactive sphingolipid PHS that inhibits autophagy.